Novel Strategies for Identification and Isolation of Murine Adult HSCs and Embryonic Pre-HSCs

Billions of blood cells are produced in the body on a daily basis. At the top of the hematopoietic hierarchy are the hematopoietic stem cells (HSCs) that are able to self-renew, give rise to all downstream progenitors, and engraft long-term upon transplantation into a conditioned recipient. HSC transplantation (HSCT) can be employed to repopulate a defective blood system with a healthy one. In fact, HSCT has the potential to treat any disease that is inherent to the blood system. Yet due to the complications associated with it, HSCT is often utilized as a last-resort option. In recent years, the advent of induced pluripotent stem cells (iPSCs) has promised a definitive solution to HSCT-related complications by introducing the prospect of patient-specific iPSC-derived HSCs (iHSCs). Encouraging advancements have produced iHSCs that exhibit HSC-like properties, however, generation of iHSCs with therapeutic potential has failed due to a lack in robust BM engraftment potential.Generated iHSCs share similar characteristics to the embryonic precursors to HSCs, pre-HSCs, which also lack BM engraftability. Understanding the natural pathways that drive maturation of pre-HSCs into BM-engraftable HSCs can shed much-needed light on the necessary signals required for the generation of therapeutic iHSCs. However, our understanding of the development of the hematopoietic system is incomplete. The ability to better isolate HSCs and pre-HSCs would allow improved characterization of these cells and highlight the molecular differences between them, and perhaps, identify the defining factors in pre-HSC maturation.Here we report the use of CD11a as a potent marker of adult HSCs and embryonic pre-HSCs. We introduce an efficient strategy for FACS-purification of adult HSCs. This new strategy addresses the issues associated with HSC sorting in conditions where conventional HSC markers fail. Furthermore, we provide evidence for the efficacy of CD11a in combination with another HSC marker, endothelial protein C receptor (EPCR; also known as CD201), for the isolation of all BM HSCs without the need for additional HSC markers. CD11a can also be used to identify embryonic pre-HSCs. Here we demonstrate that CD11a greatly improves the purity of pre-HSCs in a rare sorted population. Moreover, our sorting strategy with CD11a allows the isolation of all pre-HSCs from all the tissues and the timepoints that are relevant to pre-HSC biology. Furthermore, we confirm the identity of our putative pre-HSC population with the use of an entirely in vivo model (instead of a commonly-used ex vivo assay which may not reveal pre-HSC-specific activity). Based on our findings, we propose that improved characterization of cellular and molecular players in the developing liver niche can benefit our understanding of pre-HSC maturation. Altogether, we establish CD11a as an indispensable marker for adult HSC and embryonic pre-HSC identification and isolation

The CD11a and Endothelial Protein C Receptor Marker Combination Simplifies and Improves the Purification of Mouse Hematopoietic Stem Cells

Abstract Hematopoietic stem cells (HSCs) are the self‐renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, endothelial protein C receptor (EPCR), can be used to effectively identify and purify HSCs. We introduce a new two‐color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist's toolkit improves the purity of and simplifies isolation of HSCs. stem cells translational medicine 2018;7:468–47

Absence of CD11a Expression Identifies Embryonic Hematopoietic Stem Cell Precursors via Competitive Neonatal Transplantation Assay.

Hematopoietic stem cells (HSCs) are defined by their self-renewal, multipotency, and bone marrow (BM) engraftment abilities. How HSCs emerge during embryonic development remains unclear, but are thought to arise from hemogenic endothelium through an intermediate precursor called "pre-HSCs." Pre-HSCs have self-renewal and multipotent activity, but lack BM engraftability. They can be identified functionally by transplantation into neonatal recipients, or by in vitro co-culture with cytokines and stroma followed by transplantation into adult recipients. While pre-HSCs express markers such as Kit and CD144, a precise surface marker identity for pre-HSCs has remained elusive due to the fluctuating expression of common HSC markers during embryonic development. We have previously determined that the lack of CD11a expression distinguishes HSCs in adults as well as multipotent progenitors in the embryo. Here, we use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed "11a- eKLS" (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all in vivo long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs

Graft conditioning with fluticasone propionate reduces graft‐versus‐host disease upon allogeneic hematopoietic cell transplantation in mice

Abstract Hematopoietic cell transplantation (HCT) treats many blood conditions but remains underused due to complications such as graft‐versus‐host disease (GvHD). In GvHD, donor immune cells attack the patient, requiring powerful immunosuppressive drugs like glucocorticoids (GCs) to prevent death. In this study, we tested the hypothesis that donor cell conditioning with the glucocorticoid fluticasone propionate (FLU) prior to transplantation could increase hematopoietic stem cell (HSC) engraftment and reduce GvHD. Murine HSCs treated with FLU had increased HSC engraftment and reduced severity and incidence of GvHD after transplantation into allogeneic hosts. While most T cells died upon FLU treatment, donor T cells repopulated in the hosts and appeared less inflammatory and alloreactive. Regulatory T cells (Tregs) are immunomodulatory and survived FLU treatment, resulting in an increased ratio of Tregs to conventional T cells. Our results implicate an important role for Tregs in maintaining allogeneic tolerance in FLU‐treated grafts and suggest a therapeutic strategy of pre‐treating donor cells (and not the patients directly) with GCs to simultaneously enhance engraftment and reduce GvHD upon allogeneic HCT

Graft conditioning with fluticasone propionate reduces graft‐versus‐host disease upon allogeneic hematopoietic cell transplantation in mice

Hematopoietic cell transplantation (HCT) treats many blood conditions but remains underused due to complications such as graft-versus-host disease (GvHD). In GvHD, donor immune cells attack the patient, requiring powerful immunosuppressive drugs like glucocorticoids (GCs) to prevent death. In this study, we tested the hypothesis that donor cell conditioning with the glucocorticoid fluticasone propionate (FLU) prior to transplantation could increase hematopoietic stem cell (HSC) engraftment and reduce GvHD. Murine HSCs treated with FLU had increased HSC engraftment and reduced severity and incidence of GvHD after transplantation into allogeneic hosts. While most T cells died upon FLU treatment, donor T cells repopulated in the hosts and appeared less inflammatory and alloreactive. Regulatory T cells (Tregs) are immunomodulatory and survived FLU treatment, resulting in an increased ratio of Tregs to conventional T cells. Our results implicate an important role for Tregs in maintaining allogeneic tolerance in FLU-treated grafts and suggest a therapeutic strategy of pre-treating donor cells (and not the patients directly) with GCs to simultaneously enhance engraftment and reduce GvHD upon allogeneic HCT

The adaptive immune system restrains Alzheimer's disease pathogenesis by modulating microglial function.

The innate immune system is strongly implicated in the pathogenesis of Alzheimer's disease (AD). In contrast, the role of adaptive immunity in AD remains largely unknown. However, numerous clinical trials are testing vaccination strategies for AD, suggesting that T and B cells play a pivotal role in this disease. To test the hypothesis that adaptive immunity influences AD pathogenesis, we generated an immune-deficient AD mouse model that lacks T, B, and natural killer (NK) cells. The resulting "Rag-5xfAD" mice exhibit a greater than twofold increase in β-amyloid (Aβ) pathology. Gene expression analysis of the brain implicates altered innate and adaptive immune pathways, including changes in cytokine/chemokine signaling and decreased Ig-mediated processes. Neuroinflammation is also greatly exacerbated in Rag-5xfAD mice as indicated by a shift in microglial phenotype, increased cytokine production, and reduced phagocytic capacity. In contrast, immune-intact 5xfAD mice exhibit elevated levels of nonamyloid reactive IgGs in association with microglia, and treatment of Rag-5xfAD mice or microglial cells with preimmune IgG enhances Aβ clearance. Last, we performed bone marrow transplantation studies in Rag-5xfAD mice, revealing that replacement of these missing adaptive immune populations can dramatically reduce AD pathology. Taken together, these data strongly suggest that adaptive immune cell populations play an important role in restraining AD pathology. In contrast, depletion of B cells and their appropriate activation by T cells leads to a loss of adaptive-innate immunity cross talk and accelerated disease progression

The adaptive immune system restrains Alzheimer's disease pathogenesis by modulating microglial function.

The innate immune system is strongly implicated in the pathogenesis of Alzheimer's disease (AD). In contrast, the role of adaptive immunity in AD remains largely unknown. However, numerous clinical trials are testing vaccination strategies for AD, suggesting that T and B cells play a pivotal role in this disease. To test the hypothesis that adaptive immunity influences AD pathogenesis, we generated an immune-deficient AD mouse model that lacks T, B, and natural killer (NK) cells. The resulting "Rag-5xfAD" mice exhibit a greater than twofold increase in β-amyloid (Aβ) pathology. Gene expression analysis of the brain implicates altered innate and adaptive immune pathways, including changes in cytokine/chemokine signaling and decreased Ig-mediated processes. Neuroinflammation is also greatly exacerbated in Rag-5xfAD mice as indicated by a shift in microglial phenotype, increased cytokine production, and reduced phagocytic capacity. In contrast, immune-intact 5xfAD mice exhibit elevated levels of nonamyloid reactive IgGs in association with microglia, and treatment of Rag-5xfAD mice or microglial cells with preimmune IgG enhances Aβ clearance. Last, we performed bone marrow transplantation studies in Rag-5xfAD mice, revealing that replacement of these missing adaptive immune populations can dramatically reduce AD pathology. Taken together, these data strongly suggest that adaptive immune cell populations play an important role in restraining AD pathology. In contrast, depletion of B cells and their appropriate activation by T cells leads to a loss of adaptive-innate immunity cross talk and accelerated disease progression

The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function

The innate immune system is strongly implicated in the pathogenesis of Alzheimer’s disease (AD). In contrast, the role of adaptive immunity in AD remains largely unknown. However, numerous clinical trials are testing vaccination strategies for AD, suggesting that T and B cells play a pivotal role in this disease. To test the hypothesis that adaptive immunity influences AD pathogenesis, we generated an immune-deficient AD mouse model that lacks T, B, and natural killer (NK) cells. The resulting “Rag-5xfAD” mice exhibit a greater than twofold increase in β-amyloid (Aβ) pathology. Gene expression analysis of the brain implicates altered innate and adaptive immune pathways, including changes in cytokine/chemokine signaling and decreased Ig-mediated processes. Neuroinflammation is also greatly exacerbated in Rag-5xfAD mice as indicated by a shift in microglial phenotype, increased cytokine production, and reduced phagocytic capacity. In contrast, immune-intact 5xfAD mice exhibit elevated levels of nonamyloid reactive IgGs in association with microglia, and treatment of Rag-5xfAD mice or microglial cells with preimmune IgG enhances Aβ clearance. Last, we performed bone marrow transplantation studies in Rag-5xfAD mice, revealing that replacement of these missing adaptive immune populations can dramatically reduce AD pathology. Taken together, these data strongly suggest that adaptive immune cell populations play an important role in restraining AD pathology. In contrast, depletion of B cells and their appropriate activation by T cells leads to a loss of adaptive–innate immunity cross talk and accelerated disease progression

Identification of IL-40, a Novel B Cell-Associated Cytokine.

We describe a novel B cell-associated cytokine, encoded by an uncharacterized gene (C17orf99; chromosome 17 open reading frame 99), that is expressed in bone marrow and fetal liver and whose expression is also induced in peripheral B cells upon activation. C17orf99 is only present in mammalian genomes, and it encodes a small (∼27-kDa) secreted protein unrelated to other cytokine families, suggesting a function in mammalian immune responses. Accordingly, C17orf99 expression is induced in the mammary gland upon the onset of lactation, and a C17orf99-/- mouse exhibits reduced levels of IgA in the serum, gut, feces, and lactating mammary gland. C17orf99-/- mice have smaller and fewer Peyer's patches and lower numbers of IgA-secreting cells. The microbiome of C17orf99-/- mice exhibits altered composition, likely a consequence of the reduced levels of IgA in the gut. Although naive B cells can express C17orf99 upon activation, their production increases following culture with various cytokines, including IL-4 and TGF-β1, suggesting that differentiation can result in the expansion of C17orf99-producing B cells during some immune responses. Taken together, these observations indicate that C17orf99 encodes a novel B cell-associated cytokine, which we have called IL-40, that plays an important role in humoral immune responses and may also play a role in B cell development. Importantly, IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observations suggest that it may play a role in the pathogenesis of certain human diseases